MICROBIOLOGICAL PROBLEMS MEATSvarious bacteria which can bring about these oxidizing reactions are:...

MICROBIOLOGICAL PROBLEMS IN THE PRESERVATION OF MEATS

L. B. JENSENResearch Laboratories, Swift & Company, Chicago, Illinois

CONTENTS

1. Action of Certain Bacteria on Myohemoglobin and Nitric-Oxide Derivatives Formedin Cured Meats................................................................ 162

2. Ante-Mortem'and Post-Mortem Changes of Tissues of Food Animals..164(a) Bacteriology of Tissues of Living and Slaughtered Hogs.................... 164(b) Autolysis of Tissues........................................................ 166

3. Action of Micro6rganisms on Fats ............................ 1694. Effect of Nitrates and Nitrites on Bacteria in Cured Meats ................ 170

(a) Action of Nitrates......................................................... 170(b) Action of Nitrites.......................................................... 173(c) Action of Nitrates on Gas Formation by Species of Bacillus ............. 174

5. Action of Salt (NaCI) on Bacteria ............................ 175(a) Action of Salt on Bacteria in Meats........................................ 175(b) Effect of Impurities in Salt ............................. 177(c) Bacteria in Salt .................................. 178(d) Some Effects of Salt on Bacteria .......................... 179

6. Some Recent Developments in Meat Technology ..................... 180(a) Dehydration of Meats...................................................... 180(b) Bacterial Standards .................. 182(c) Hams .................. 183(d) Control of Spores in Canned Meats................................. 183

7. References ................................. 184

Until some sixty years ago, man depended largely upon the perishable foodsupplies produced within a few hundred miles of his home. In general, themethods of preparation and the kinds of foodstuffs of animal origin had changedlittle since ancient times. With the rise of bacteriology, coupled with develop-ments in the physical and chemical sciences, procedures have been elaborated forthe control and study of meat foods. However, the pace of all historical develop-ment teaches us that the work in meat technology, as in other fields of foodtechnology, has just begun.For the preservation of meats, the following practical methods have been de-

veloped, some of them with an empirical background of great antiquity: partialdrying and dehydration; curing with salt, nitrates, nitrites, and sugars; smoking,icing and refrigeration; culinary heating; packing in metal and glass containers.The maintenance of good standards of quality as well as of sanitation in theproducts often depends on adequate microbiological control; and, on occasion,specific microbiological problems are presented which depend, for an ultimatesolution, upon fundamental research.

Practically every process employed in the meat industry hinges upon the re-duction and control of microbiological activities. The proteins, carbohydratesand fats of meat foods are all vulnerable to the action of many kinds of micro-organisms. There are numerous species of nonpathogenic bacteria, yeasts, and

161

on June 13, 2020 by guesthttp://m

mbr.asm

.org/D

ownloaded from

http://mmbr.asm.org/

L. B. JENSEN

molds which can discolor fresh and cured meats by oxidative processes; induceoxidative rancidity in fats, and discolor fats and oils with their pigments; fermentcured meats and canned meats with carbon dioxide production; and cause spoil-age of unprotected proteins in a number of ways. For discussion of the manyactivities of micro6rganisms in meat foods and practical measures for control ofmicro6rganisms in meat plants, the reader is referred elsewhere (36).The following sections illustrate a few of the problems arising from large-scale

processing of meat foods which may be of general bacteriological interest.

1. ACTION OF CERTAIN BACTERIA ON MYOHEMOGLOBIN AND NITRIC-OXIDEDERIVATIVES FORMED IN CURED MEATS

The color of muscle tissue is red in the fresh, unheated state, but this color isnot due to residual blood or hemoglobin. The pigment is myohemoglobin (ormyoglobin), an integral part of the muscle tissue, which does not circulate in theblood stream. Hektoen, Robscheit-Robbins, and Whipple (27), by means of theprecipitin reaction, have demonstrated the non-identity of myoglobin and hemo-globin. However, the general properties of these two pigments are sufficientlyalike so that one may use the blood pigment for certain experiments in place of themuscle pigment without serious error in the conclusions drawn therefrom, ac-cording to Urbain and Jensen (103).

Unlike fresh meats, the cured meats retain their redness on cooking. Chieflythrough the work of Haldane (25) and Hoagland (29), the stability of the curedmeat pigment towards heat has been shown to be a property of nitric-oxidederivatives of the pigment resulting from the chemical interaction with nitriteeither added directly in the curing salts or produced through bacterial reductionof nitrate. (These investigators assumed the pigment of meat to be identicalwith the blood pigment.) Successful curing of the meat eventually leads to theformation of nitric-oxide myohemoglobin, according to the reaction: NO + myo-hemoglobin = nitric-oxide myohemoglobin. The source of the nitric oxide isnitrite or more properly nitrous acid. Upon heating, the nitric-oxide myohemo-globin is converted to nitric-oxide hemochromogen, a denatured protein. Thefact that this hemochromogen is red is one of the objects gained by the employ-ment of nitrite and nitrate in the curing of meats. Any reaction which yieldsunusual colors in cured meats is undesirable as a matter of custom rather than ofsanitary deficiency.

In the past, one of the most troublesome phenomena in the meat plant was thediscoloration of cured meats, such as green rings in frankfurters, green boiledhams, and dried beef hams. The pink nitric-oxide pigments are sensitive to oxi-dations of microbial origin and become emerald green, gray, and brown in colorwhen "oxidase"-forming bacteria act upon them. Reducing agents can oftenrestore the pink color but the use of compounds, such as sulfites, is prohibited bylaw. The greening action of a few species of bacteria on blood agar media is socharacteristic that considerable diagnostic importance has been attached to thephenomenon. The green pigment formed has been ascribed to a variety of

162


mbr.asm

.org/D

ownloaded from


MICROBIOLOGY AND PRESERVATION OF MEATS

causes, among which are the following: formation of methemoglobin, Zinsserand Bayne-Jones (115), and Valentine (104); action of lactic acid, Ruediger (87)and Hagan (24); oxidation, McLeod and Gordon (71); reduction, Holman (31);xanthroproteic reaction, Barnard and Gowen (4); and an optical illusion, Boxer(7).Jensen and Urbain (51) have shown that many species of bacteria possess

mechanisms for discoloring blood pigments and their nitric-oxide derivatives.They found that the kinds of microorganisms responsible for the formation ofgreen pigments in meats and blood-agar are those which oxidize hemoglobin,nitric-oxide hemoglobin, nitric-oxide hemochromogen, and hematin. In ad-dition, bacteria producing hydrogen sulfide cause green discolorations of thesepigments. The role of hydrogen sulfide in the production of these discolorationsis one that is somewhat involved. Hydrogen sulfide reacts with reduced hemo-globin to form a purple compound. On exposure to oxygen, this purple com-pound oxidizes rapidly to form a green compound. Spectrophotometric measure-ments have been made on the green pigments obtained by the action of certainoxidizing bacterial enzymes on nitric-oxide hemoglobin and by the action ofhydrogen sulfide on hemoglobin. These compounds were found to be spectro-scopically different from methemoglobin. It will be remembered that methemo-globin is brown in color, not green. Hence, in speaking of the greening action ofsome bacteria on blood-agar plates, one should not designate the greenish halos asdue to methemoglobin. Recent work indicates that these green pigments or"verdohemes" may be related to, or identical with, some of the bile pigments likebiliverdin. The reader is referred to the publications of Lemberg and col-laborators (61 to 67) for information on the chemistry and occurrence of some ofthese green pigments in various biological materials.Green pigments, prepared by treating fresh ground beef myoglobin and fresh

defibrinated hog's blood with H202 and NaNO2 and also with H2S, were fed to 25white rats and 25 white mice, supplementing 20% of the green pigments in thestandard basal diet. Each lot of test animals was fed these pigments for onemonth with no ill effects. These test animals were irradiated each week for 30minutes under a General Electric S2 ultraviolet lamp; and they exhibited no un-toward reactions to this treatment, i.e., there was no photo-sensitization inducedby porphyrins as described by Blum (6).What are commonly spoken of as "green rings" in sausage occur near the

casing, varying in frankfurters, for example, in distance from the casing from one-sixteenth to one-eighth of an inch (1.5 to 3 mm), and in width from a faint-attimes, discontinuous-circle to a band one-eighth of an inch (3 mm) across.The green discoloration mayappear also as a core in the center of the sausage. Thecircular form of green ring has often been noted in square-pressed bologna. Thevarious bacteria which can bring about these oxidizing reactions are: (a) thosewhich elaborate oxidizing enzymes, some insensitive to catalase and thermostable,and some sensitive to catalase and thermolabile (effects destroyed by heating to155-160F (68.4-71.1C) for 15 minutes); (b) H2S-forming bacteria, both aerobes

163


mbr.asm

.org/D

ownloaded from


L. B. JENSEN

and facultative anaerobes growing at an optimum zone in the sausage; (c) "oxi-dizing" bacteria growing at an optimum zone only. The ring formation isprobably due to a phenomenon similar to that observed by Neill and Hastings(76) in which the oxygen tension governs the amount of oxidized hemoglobin inthe presence of a constant amount of pneumococcus extract. Their data showthat when the oxygen tension approaches zero, only traces of the oxidized pig-ment are formed by the extract; and when the tension of molecular oxygen isgreat, again only traces of the oxidized pigment are formed. However, at inter-mediate, optimum oxygen tensions much oxidized pigment is formed. In manyinstances, the green-ring formation in casing sausage is formed according to thequantity of oxygen dissolved, provided that the bacteria have grown sufficientlyin the trimmings from which the sausage was made. The addition of sterilebacterial filtrates to fresh sausage materials has resulted in the production ofgreen rings in the finished product. For a detailed discussion of these green rings,the reader is referred elsewhere (38).The iridescence of sliced cured meats, such as ham, bacon, and dried beef, is

due to the peculiar surfaces of cured meat fibers. The color is a structural coloras opposed to a pigment color. It is due to the breaking up of white light by thehighly fibrous character of the surface and to the film of fat on these fibers. Ifthe fat is removed, the iridescence disappears. Any oil applied to the defattedsurface will restore iridescence. The phenomenon has no sanitary significance,and is related to that of the diffraction grating or "clam-shell" play of colors.

2. ANTE-MORTEM AND POST-MORTEM CHANGES OF TISSUES OF FOOD ANIMALS

(a) Bacteriology of Tissues of Living and Slaughtered Hogs. There is very littleinformation available as to the occurrence of bacteria in tissues and blood inhealthy persons, according to Topley and Wilson (100). However, some writers(Norris and Pappenheimer (77), Arnold (3), and Burn (11) ) state that there isdefinite evidence that tissues may not always be sterile in normal human beings.Most of the early work on the bacteriology of tissues of normal animals indicatesthat tissues, especially muscle, are sterile. Other workers have reported thepresence of a variety of micro6rganisms in the blood, viscera, and muscular tis-sues of healthy animals (48). In 1926 Reith (82), after a careful study of varioustissues of hogs and laboratory animals, came to the conclusion that aerobes andanaerobes are present in the musculature and blood of apparently normal animals.If one upholds the ante-mortem infection theory, the problem of meat spoilagebecomes a refrigeration-engineering problem to a large extent, and whateverspoilage occurs must be considered unavoidable even though applied bacteriologyfunctions ideally. However, the work of Burn (11) on post-mortem bacteriologypoints to the phenomenon of agonal invasion; and the work of Jensen and Hess(485 supports the view that the invasion of bacteria is agonal and post-mortemrather than ante-mortem. A series of biopsy studies was conducted on primenormal hogs whose blood, bones, bone marrows, and muscle tissues were examinedbacteriologically. After the surgical fields were closed, sutured, and heavilycovered with celloidin, the animals were immediately taken to the killing floor,

164


mbr.asm

.org/D

ownloaded from



hoisted, stuck and bled, washed, dehaired, butchered, and dressed. Before theywere chilled and tanked, the control tissues and expressed blood were examined(40 minutes later). Except for one animal (which appeared normal, but harboredHemophilus sp. in all tissues examined while alive and after dressing as well),none of the hogs, while alive, showed the presence of microorganisms in the tissuesor the blood. The post-mortem findings indicated, however, that bacteria mayoccasionally be found in the tissues and blood. The tissues post-mortem mayshow the presence of species of Achromobacter, Pseudomonas, Serratia, Bacillus,Proteus, Micrococcus, Clostridium, "diphtheroids," and Torula (48). These find-ings suggest that bacteria may on occasion be found in tissues immediately afterdeath from slaughter.Many investigators have determined a few of the mechanisms which remove

bacteria from the blood stream, a subject which has been reviewed elsewhere (48).Norris and Pappenheimer (77), who introduced Serratia marcescens into the oralcavity of human beings immediately after death, recovered these bacteria fromthe lungs several hours later. Some pathologists say that contamination of theheart's blood occurs through the large veins from the lungs, and that heart'sblood at autopsy practically always shows contamination with many species ofmicro6rganisms. It has long been known by pathologists that, at autopsy,bacteria and remnants of food may be found in the lungs, i.e., "food down thewrong way." It is not uncommon to find corn and other food in the lungs ofslaughtered hogs. Huilphers (33) describes micro6rganisms found in lungs ofslaughtered hogs, and most of these micro6rganisms appear to be soil flora.There is a unique mechanism of contamination of some living animals shown firstby Tarozzi (96) and later by Canfora (12). Each of them injected spores ofClostridium tetani into animals. There were no ill effects from these inoculations.After a considerable length of time (up to 55 days), a bone of one of the animalswas broken or traumatized, following which tetanus usually occurred, showing thelongevity of spores in the normal animal body. Koser and McClelland (56)found the spores of clostridia recoverable from the body long after the spores ofaerobes had disappeared. Contamination of the bone marrows, especially thered marrows, may perhaps occur in the normal animal through permeability ofthe intestinal mucosa, via wounds, the lungs, and the upper respiratory tract.In this connection, the occurrence of latent infections should also be considered,Meyer (73).Many theories, current in the meat-processing industry, on the source of con-

tamination of tissues have been investigated (48), and brief mention is made ofsome experiments which may be deserving of further study. The skin of a hogobviously is heavily contaminated with micro6rganisms. When the stick knifepasses through the skin, severing the jugular vein and sometimes the carotidartery, the blade is washed with venous and often with arterial blood. The heartmay beat from two to nine minutes after the stick wound is made. Some of theshackled, hoisted hogs contract their heads in the direction of their forelegs andthus withhold some of the blood by constriction and hematoma, allowing someblood from this area to reach the heart and be circulated. A "negative" pressure

165


mbr.asm

.org/D

ownloaded from


L. B. JENSEN

may be set up in the severed or pierced vessels, owing to the labored breathingaccompanying exsanguination (oxygen starvation). The flow of the pooled bloodand blood within the vein is towards the heart.

Tests were made by dipping the blades in cultures of various bacteria and thensticking the hogs in the usual manner. Some of these bacteria (possessing anidentifying cultural characteristic to distinguish them from the usual flora) wererecovered from the marrow of the tibia and other bones. A large number of hogswere then bled aseptically (a technic which obviously brings to mind the methodsemployed by the serum companies which produce hog-cholera serum and.otherimmunizing serums. It has long been observed that such "serum" hogs whenslaughtered produce a very low incidence of sour meats). However, the asepticbleeding as effected in the above tests did not close all portals of contaminationof the tissues, including bone marrow; but technical sanitation of the pork-blockin conjunction with other methods of applied bacteriology, reviewed elsewhere(48), effected a marked reduction in the numbers of sour marrows and tissues.

Practically all investigators in this field have observed the absence of coliformbacteria in hams. Boyer (8) states: "The absence of the Bacillus coli group oforganisms from the numerous cultures taken from these hams is of particularinterest. The members of this group are abundant and ubiquitous on the killingfloor, and are almost invariably found on the surfaces of the carcasses which areexposed during killing floor operations. Their absence is of special significancein that it goes far to eliminate the possibility that organisms present in the hamsgain access during killing floor operations."The stick-blade contamination and the train of conditions following the sever-

ing of the neck vessels should favor the entry of coliform and other bacteria intothe blood and marrow. However, some factor prevents their reaching the mar-row or surviving therein if they do reach it. It is well known that undiluted,fresh mammalian blood is bactericidal (1). Hog's blood was drawn asepticallyfrom the tail of a live hog, as is done by the commercial producers of serum, andbled directly into sterile containers containing glass beads for defibrination.Various freshly isolated strains of bacteria (Escherichia coli, species of Pseu-domonas, Serratia, Achromobacter, vegetative clostridia and Bacillus, and Staphy-lococcus aureus) were added in minimum amounts of menstruum so that the bloodwould contain about 50,000 viable cells per milliliter. The defibrinated bloodcontaining these bacteria was cultured at short intervals up to 24 hours. It wasfound that many of the flasks were practically sterile after 2 to 5 hours and otherstrains survived with less complete reduction in numbers. The ham-souringtypes of bacteria, such as certain strains of Serratia, Achromobacter, Clostridiumputrefaciens of McBryde, and Pseudomonas, were more or less resistant to thebactericidal effects of hog's blood. The suspensions of Staphylococcus aureus andEscherichia coli were often sterile after 2 to 5 hours. These studies should be re-peated and extended.

(b) Autolysis of Tissues. Over 70 years ago Hoppe-Seyler (32) recognizedliquefaction of dead tissues occurrnmg without accompanying putrefaction, and henoted that the phenomenon resembled the effects of digestive ferments. Some

166


mbr.asm

.org/D

ownloaded from



experiments performed by Ernst Salkowski (88) in 1890 led this investigator tothe discovery of tissue autolysis, which he named "autodigestion." He usedchloroform as a bacteriostat. Little notice was taken of this work for a decadeuntil Martin Jacoby (35) took up the work again and introduced the term"autolysis."The one great difficulty in evaluating true autolysis is the uncertainty of con-

tinuing and effective antisepsis. Von Furth (107) states that it was assumedthere would be no danger of bacterial growth in finely divided tissues saturatedwith chloroform or toluol. He states that it was usually supposed that, if a fewdrops of toluol or a few bits of thymol were added to a thick emulsion of sometissue, it would be quite safe to incubate the emulsion for months, and that thedanger of bacterial action "would be surely and for all time eliminated by such apurely symbolic performance (for that is actually about all it represents)....It is quite obvious why 'discoveries' prospered and multiplied in our literature onthe same scale as did the bacteria in the pots and jars of the experimenters."The question of tissue sterility also enters into the picture. There are times

when many tissues of slaughtered animals are not sterile, although biopsy ma-terials are usually sterile.A large number of experiments have been performed to demonstrate the native

autolytic enzymes of tissues (109). Some microbiologists do not subscribe to theexistence of autolytic enzymes because of the faulty bacteriological techniquesemployed in certain biochemical experimentation. Recent work by Reeves andMartin (81) demonstrates that bacteria do grow in many tissue preparations con-taining preservatives calculated to stop bacterial growth. In their experiments,the sterility of digests could be determined only by smears and cultures. Theyrepeatedly found bacteria growing in digests which were free from putrid odorsand showed no evidence of bacterial growth. However, there are methods (2)available for the isolation of autolytic enzymes. Further work is needed in thisfield with new preservatives and more adequate microbiological controls.

There are several opinions extant on the exact mechanisms responsible for thetendering of meat. Some observers believe that the connective tissues are re-sponsible for tough meat and that the change from collagen (of connective tissues)to gelatin correlates closely with the degree of tenderization. Other workershave concluded that connective tissues are very little changed and that thetenderness of meat is due to the muscle fibers becoming inelastic and thus tender.Another group does not consider that autolysis by native ferments plays any ap-preciable role in meat tendering during the first month of storage and does notreckon that micro6rganisms aid in the process.The question of autolysis has been discussed in detail by Hoagland, McBryde,

and Powick (30) who believe that meat tendering during storage may be regardedas largely due to enzyme action. There is no question that some denaturation ofsoluble protein occurs during rigor mortis. Denaturation, as well as autolysis ofmuscle, favors subsequent bacterial decomposition of muscle. Gibbons andReed (21) studied the effects of autolysis on the subsequent bacterial attack in themuscle and kidney tissues of haddock. They found that the degree of autolysis,

167


mbr.asm

.org/D

ownloaded from


L. B. JENSEN

preceding the introduction of bacteria, did not affect the growth rate but didmake a marked difference in the subsequent chemical changes.

Ripening and tendering beef by aging in coolers with controlled humidities andtemperatures is the common method employed in peace times in large meatplants. During the holding period, molds of several genera appear on the cutsurfaces of the meat (usually, species of Thamnidium, Rhizopus, and Mucor).The bacteria found on the surfaces are usually species of Achromobacter andPseudomonas. Hundreds of examinations of "whiskery" beef stored for tender-ing have revealed this flora. There is a sharp difference of opinion on thequestion whether or not micro6rganisms aid in producing the organoleptic quali-ties demanded of aged beef or whether autolysis produces these effects.Under ordinary conditions beef is held about 5 days at 360 to 38°F (2.20 to

3.30C) after slaughter before it is removed to various departments for finaldisposition. If the beef is to be held for "ripening" so that increased tendernessand flavor develops, it is observed that at higher temperatures shorter holdingperiods are required. For instance, beef held at the following temperatures andtimes show practically the same degree of tenderness (as judged by experts):

21 days at 34°F ( 1.1°C)8 days at 40°F ( 4.40C)5 days at 47°F ( 8.3°C)3 days at 60°F (15.6°C)

At the expiration of these holding periods at the various temperatures the meatshould be handled promptly. There is no noteworthy difference in flavor of thelean meat, of the fat, or in the juiciness of the comparable steaks.The prevention of shrink, preservation of bloom, and control of microorganisms

on meats are subjects on which experiments go on continuously. The student isreferred to the work of A. W. Ewell (15-18) for the practical effects of ultravioletlight and ozone on beef and foods in refrigerators of high humidity.Many investigations have been made on the tendering effect of enzymes on

sausage casings, ground meats, and other protein foods. Many enzymes havebeen used experimentally for this purpose. The enzyme of the osage orange(macin) is a strong protease, as is the asclepian from the juice of the milkweed.The action of the protease of edible mushrooms has long been observed when fry-ing meats with fresh edible mushrooms. Ficin, the rapid acting protease of figs,can be used; and bromelin, the strong proteolytic enzyme in fresh pineapple juice,has been used for some time in tendering casings of frankfurters (80). Papain,the strong proteolytic enzyme of papaya, has been used in the Americas for a longtime, Kilmer (52); and the proteolytic enzyme of the Aspergillus oryzae-Jkwusgroup can be used for any tenderizing of foods. None of these preparations canbe used in inspected plants without permission of the Federal Meat InspectionDivision.The temperature at which such an enzyme becomes inactivated is very im-

portant; the ideal preparations are those inactivated at about 170F (77C). If the

168


mbr.asm

.org/D

ownloaded from



enzyme is not inactivated at 165 to 175F (74 to 79.5C) during culinary heatingthe meat tends to become mushy or butyrous in texture which is organolepticallyundesirable.Enzymes are not used in tenderizing hams or in the preparation of ready-to-eat

hams; to accomplish this end, the ready-to-eat hams are cooked in the smoke-house, and tenderized hams are heated in the smokehouse to an inside tempera-ture of 137F (58.4C) or higher.

3. ACTION OF MICROORGANISMS ON FATS

The action of microorganisms on oils and fats (other than butter fat) is a fieldawaiting extended study. Bacterial and mold metabolism of proteins and theirderivatives and of carbohydrates have been studied extensively with brilliantresults; but in the case of fat metabolism greater difficulties are encountered bothin the few varieties of microorganisms that will grow on such substrates and thecharacter of the split products. Again, many of the mechanisms of oil and fat"spoilage" have not generally been considered by oil chemists to be due tomicrobial action. To be sure, pure refined fats and oils freed from tissues andmoisture are more prone to attack from atmospheric oxygen, light, and activecatalysts like copper or iron than to direct contact with subsequent growth ofmicroorganisms; but it has been proved in large-scale tests that fats, producedfrom animal and vegetable tissues that have been handled under the best sani-tary conditions, show greater stability than fats processed from tissues showinglarge numbers of lipolytic and oxidizing micro6rganisms.Cohn H. Lea, who has done extensive work on this subject, states (59): "It is

only comparatively recently that oxidizing enzymes have come to be regarded aspotential accelerators of oxidative rancidity in foodstuffs. The earliest work ap-pears to have been that of Jensen and Grettie (44), who inoculated fats withoxidase-producing organisms and found that the development of rancidity wasfrequently accelerated. The method they used for identifying oxidase-formerswas to plate out the mixed organisms onto fat-emulsion agar and, after incuba-tion, to flood the surface of the medium either with dimethyl-p-phenylenediamine,or with the Nadi reagent. Colonies of oxidase-producing organisms treated inthis way stained red or violet-blue, owing to oxidation of the reagent to a highlycolored quinonoid compound. More recently similar colour reactions have beenobtained from the soya bean and from pork adipose tissue, indicating that thesetoo contain enzymic oxidizing systems. Such colour tests, however, only affordevidence of general oxidizing conditions, since hydrogen peroxide produced as aby-product of any enzymic oxidation would, in the presence of peroxidase, oxi-dize p-phenylenediamine and the Nadi reagent. It is, of course, quite possiblethat fat also might be oxidized by nascent hydrogen peroxide under these con-ditions."There seems little doubt, however, that certain types of micro-organisms do

accelerate the production of oxidative rancidity in fats. Cases have been ob-served, for example, where beef fat stored under conditions favourable to the

169


mbr.asm

.org/D

ownloaded from


L. B. JENSEN

growth of micro-organisms has rapidly lost its induction period (determined afterextraction from the tissue), though separate experiment had indicated that theactivity of the tissue oxidase was probably too low to account for this result."Thoroughly dry, pure fats are incapable of supporting bacterial growth. How-

ever, when fats contain over 0.3% moisture it has been observed that micro-organisms can grow on them. The constituents of fats other than glyceridesmay be important in rancidity. Jensen and Grettie (45) state: "Oils and fats fromnatural sources contain besides fatty glycerides small amounts of other materials,such as high molecular weight alcohols, hydrocarbons, protein residues and othernitrogenous matter, phosphatides and carotenoid pigments, which cannot becompletely removed by any degree of refinement. Many of these minor con-stituents enter into and affect the reactions contributing to spoilage regardless ofthe source of the oil or fat. Their presence tends to confuse the study of thespoilage of oils and fats because they not only may decompose to small amounts ofodoriferous substances but may act as catalysts, bacterial substrates, or inhibitorseither retarding or promoting reactions contributing to the spoilage of oils andfats. "

These writers conclude that microorganisms may cause (1) oxidative rancidity,(2) hydrolysis, (3) tallowiness, and (4) flavor changes owing to the production ofvarious volatile products. It appears, therefore, that microbially induced ran-cidity is more extensive than has been realized.The literature in this field, and the present status of the problems may be

found in C. H. Lea's "Rancidity in Edible Fats," 1938 (58), H. K. Dean's"Utilization of Fats," 1938 (14), and L. B. Jensen's "Microbiology of Meats,"1942 (40).

4. EFFECT OF NITRATES AND NITRITES ON BACTERIA IN CURED MEATS

(a) Action of Nitrates. In the field of the microbiology of meats, there are fewsubjects over which there has been more controversy than on the effect of sodium,nitrate on anaerobic bacteria in meats. Nitrate has for many decades been usedin limited amounts together with sodium chloride to "cure" meats, both in piecesand comminuted. Nitrate is reduced by microorganisms in part to nitrite whichforms by chemical action the heat-stable, nitric-oxide derivatives of the meatpigment, myohemoglobin. Since 1925, the Bureau of Animal Industry (nowMeat Inspection Division) has permitted the use of sodium nitrite per se (notover 200 ppm) in meat-curing mixtures. It has been found, however, that nitrateis needed in the curing mixture and that there is much merit in the use of bothnitrates and nitrites.

In 1907 Richardson (83) pointed out that "the most beneficial effect of nitratein the curing of meat is its transformation of what would, otherwise, be anaerobicconditions, into aerobic ones in the bacteriologic sense.... I need only say herethat it has been shown that aerobic bacteria will develop in the absence of air ifnitrate is present in the culture medium and the anaerobes refuse to grow in theabsence of air and presence of nitrate. The fact that typical putrefaction(Fiiulnis) is an anaerobic process and that this process can be transformed into an

170


mbr.asm

.org/D

ownloaded from



aerobic one by the presence of saltpeter, is a most important point in the curingof meat."

Grindley, MacNeal, and Kerr (23), in an extensive research on the effect ofnitrate on bacteria in curing meats, found that in acid solutions (meats areusually at pH 5.9 to 6.1) even small amounts of nitrate exerted a markedinhibitory effect on bacteria in the curing vat. The growth of salt-tolerantbacteria was restricted by small amounts of nitrate in the pickling brine.Summarizing his own work, Tanner (94) wrote: "While meat packers desired a

nitrate cure a few years ago, results of extensive investigations indicate that, attimes, a mixed cure (containing both sodium nitrate and sodium nitrite) isdesirable from the standpoint of both spoilage and development of toxin byClostridium botulinum. The preserving effects of such curing solutions have beengenerally attributed to the nitrites, either those added as such or those securedby reduction of the nitrate; however, evidence has been recently collected whichmight indicate that the nitrate itself may have some value as a preservative.This position is suggested by the fact that curing solutions containing nitratehave shown preservative action even though the nitrate reducers have beendestroyed by the process."

Large-scale tests were performed in the Swift & Company Research Labora-tories (36) on the effect of sodium nitrate' on spiced ham containing approxi-mately 10,000 spores of Clostridium sporogenes per gram in 6-pound cans. Thecans, and lots without nitrate added, were then processed for 5 hours at 68.3C(155F) according to a schedule no longer in use. The experiments showed thatnitrate is of benefit in preventing the growth of Clostridium sporogenes. It wasalso observed that spores of some species of Clostridium are killed at lower tem-peratures and at shorter holding periods when heated in the presence of 0.1%NaNO3. This adjuvant effect of nitrate was investigated by Yesair and Cameron(114) who found that the effect of curing salts on the thermal death time of sporesof the test organism is apparently to reduce the time at temperatures lower than230 to 235F (110 to 113C), and the slopes of the thermal death-time curves aregreater when the salts are added. This phase of the subject is deserving offurther study. When spores of Clostridium botulinum are incorporated in curedmeat and thermal death-times compared with those of the spores incorporated inuncured meat, the inhibitive effects of the curing salts are apparent, but when theheated meat is subcultured in a liquid medium the spores are shown to be viable.Resistance values determined by subculture after heating are approximately thesame in the cured and uncured meats. One of the shortcomings of nitrite is thatit reacts with protein when heated at canning temperatures and is destroyed, andthus if nitrate is not present the meat in cans might not be well protected fromgermination of resistant spores of clostridia.The sensitivity of species of Clostridium to oxygen still awaits satisfactory ex-

planation (91). It is held by some bacteriologists that in the absence of catalasemuch H202 can be formed by many bacteria or accumulates, and that the amount

1 To each 200 lbs. meat were added 4 oz. (0.125 %) NaNO3, 0.5 oz. (0.015 %) NaNOQ,3.5% NaCI, 2% sugar, spices.

171


mbr.asm

.org/D

ownloaded from


L. B. JENSEN

of peroxide formed might killvegetative forms of sporing anaerobes or retard theirdevelopment. This view has been objected to on the grounds that prolongedaeration of culture media containing material oxidizable by these bacteria doesnot form H202. Also when catalase is added, certain clostridia do not growaerobically. Again, it is held that washed suspensions of Clostridium sporogenesaerated in an oxidizable medium fail to take up oxygen.

Leifson (60) found, however, in extended experiments on spore-forming bac-teria, that only vegetative anaerobic cells were oxygen sensitive. These cellswere so sensitive that most of them were killed by less than 2 to 3 minutes of ex-posure to air. The change from the nonsensitive spore to the exceedingly sensi-tive vegetative cell was so abrupt that only in a few cases was the sensitivity of theintermediate stages obtained. His results also show the well-known effect ofnitrate on the germination of bacterial spores. He found 1% sodium nitrate toinhibit sporulation of Clostridium botulinum.McLeod and Gordon (72) obtained their evidence of peroxide formation by ob-

serving greening of heated blood agar (chocolate agar) a few millimeters below thesurface at a zone where penetrating atmospheric oxygen meets with growth ofclostridia. It is possible that the greening action is due to production of thiolcompounds which may, on contact with oxygen, form H202. On the other hand,H2S may form sulfhemoglobin or related compounds which, upon contact withoxygen, change into the green compounds described in Section 1.When Clostriditum botulinum is grown under anaerobic conditions on the sur-

face of blood-agar plates containing benzidine and then exposed to air, sufficientperoxide accumulates to produce dark halos. This test shows peroxide produc-tion in the presence of oxygen. Hence, since strict anaerobes apparently do notproduce catalase, the organisms are unable to destroy the toxic oxidizing com-pound. (Peroxidase does not decompose H202 in the absence of an oxidizablesubstance, whereas catalase decomposes H202 in the absence of an oxygen ac-ceptor.)The work of Hart and Anderson (26) indicates that the green discoloration pro-

duced on chocolate agar can be reproduced anaerobically by the action of reduc-ing systems, including cysteine and bacterial suspensions with hydrogen donators.The identity of the green pigment is not clear, but the above evidence is taken bymany writers to prove that hydrogen peroxide is not the only "reagent" whichcan form a green zone in blood pigments. Hart and Anderson state that dis-coloration of heated blood agar (chocolate agar) by certain streptococci is in-hibited by catalase which is direct evidence that the greening is due to H202.The greening action on unheated blood media by the pneumococci and strepto-cocci is viewed by them as an entirely different phenomenon. The discussion inSection 1 may extend some of these views.The reader is referred to the work of Broh-Kahn and Mirsky (9) who do not

agree with McLeod and Gordon's peroxide theory. They believe also that thereduction potential theory of Quastel and others is equally untenable. It maybe concluded that no theory at the present time explains why anaerobes cannotgrow in air, and in the writer's opinion, meat technologists should reserve judg-

172


mbr.asm

.org/D

ownloaded from


. MICROBIOLOGY AND PRESERVATION OF MEATS

ment as to the effect of nitrates on clostridia and the mechanisms of green-pig-ment formation until this field is more fully developed.The important work of Tarr (98) on the action of nitrite on bacteria, recently

reported, needs serious attention in evaluating the mechanisms of growth re-tardation of anaerobes and aerobes in mixed cure with nitrate.

(b) Action of Nitrites. Tarr (98) has shown that nitrites may, under certainconditions, play an important part in retarding the growth of many kinds ofbacteria and thus delay the spoilage of meats. The work done by other investi-gators has indicated that large amounts of nitrite-greatly in excess of 0.02%nitrite permitted by law-are necessary to kill or inhibit bacteria. Sinceprevious workers did not state the pH of the media they employed, it may well bethat the media were neutral or faintly alkaline. Tarr found that bacteria aresusceptible tolowconcentrations ofnitrite only at pH values below 7. Not all bac-teria studied proved equally susceptible to nitrite, and certain organisms provedto be resistant. The growth of species of the following genera at pH 5.7 to 6.0 waseither inhibited or prevented by 0.02% of sodium nitrite: Achromobacter, Flavo-bacte7ium, Pseudomonas, Micrococcus, Escherichia, Aerobacter, and one species ofTorula. Tarr found that in the acid range 0.02% NaNO2 also inhibited thegrowth of two species of obligate anaerobes, Clostridium botulinum and Clostri-dium sporogenes. Likewise, 0.02% NaNO2 inhibited the growth of Eberthellatyphosa and Staphylococcus aureus. While Tarr does not attempt to explain themechanisms of inhibition, he points out that nitrites may combine with respira-tory hematin compounds. Ingram (34) found that oxygen uptake in the case ofthe aerobic, cytochrome-containing Bacillus cereus is inhibited by traces of nitrite.There is little doubt that nitrite acts directly upon the bacterial cell, but whetherits action is general or specific is not clear, according to Tarr. He found thatNaNO2 in concentrations of 1 and 10% exerted marked bactericidal action in acidmedium, but not above pH 7. In this connection, Bittenbender et al. (5) re-ported that Staphylococcus aureus was not killed upon exposure to 38.8% ofsodium nitrite for 10 minutes at pH values between 3 and 8. These workers didnot report the numbers of organisms inhibited but recorded only the presence orabsence of growth.

Quastel and Wooldridge's (79) work suggests that nitrites inactivate certainenzymes by combining with their amino groups.The theory that inhibition of the growth of sporing anaerobes during meat-

curing processes is due to H202, a substance which is toxic for clostridia even inextremely small amounts, and which may accumulate through inhibition ofcatalase by the hydroxylamine formed in cures (46), may well account for somegrowth inhibition; but Tarr's phenomenon shows that nitrites also retardclostridia under such conditions. Some recent work has demonstrated thathydrogen peroxide does possess antibacterial action. Green and Pauli (22) haveobserved that the antibacterial action of the xanthine oxidase system is due to theformation of H202, a product of the enzymic action. Van Bruggen, Raistrick,et al. (105) have shown that penicillin B (an enzyme of flavoprotein nature) owesits bactericidal powers to H202, a product of the enzymic action. McCulloch

173


mbr.asm

.org/D

ownloaded from


L. B. JENSEN

(70) writes that H202 can prevent the growth of anaerobic organisms although itsinfluence is transitory, but it is incapable of destroying the spores of anaerobes.When nitrite in meat is heated at canning temperatures above 100C (212F),

much of the compound is destroyed. Brooks, Haines, Moran, and Pace (10)have observed that at 212F the time required for fifty per cent destruction ofnitrite increases with the initial concentration ranging from 13 to 120 minutesfor values of 30 to 589 g NaNO2 per 0l g tissue. With the usual times of cook-ing, the reduction from a high to a low nitrite content cannot be expected.The presence of nitrate in a finished sausage or other mixed-cure product on

occasion presents the disadvantage of continued production of nitrite by bacteriaduring transit to some inspection laboratory or during improper storage in thechemists' locker before undergoing analysis. Much misunderstanding has arisenfrom this avoidable bacterial action, and cured meat foods showing over 200 ppmof nitrite upon ultimate analysis have undoubtedly often left the sausage kitchenwith the lawful limit of nitrite present. It is not clear how the nitrate-reducingbacteria can grow in the presence of such large amounts of nitrite found in ex-perimentally incubated, mixed-cure sausage.

It is possible that the characteristic flavor of bacon and ham (as opposed tosalt pork) is due to a product of a reaction of nitrite with constituents of the tis-sue, either during curing or during cooking, according to Brooks, Haines, Moranand Pace (10).

(c) Action of Nitrates on Gas Formation by Species of Bacillus. The typicalgaseous swell of canned cured meats is practically always due to the fermentationof sugar by species of the genus Bacillus. Large quantities of carbon dioxide areformed when either the small or large varieties (except Donker's group) grow in amedium containing fermentable carbohydrates, nitrates, and cured pork meat,or in "sugar-cured meats" at temperatures ranging from 23.9 to 48.9C (75 to120F). This gaseous fermentation also produces spongy beef hams and causesbursts in large bologna. None of the hundreds of strains examined can form gasin the ordinary sugar broths or sugar-agar media.

Nitrate, sugar, and cured meat must be present together before the bacilliferment with gas production. If the medium contains c.p. nitrite withoutnitrate, no carbon dioxide forms. If sugar is omitted, no carbon dioxide, or verylittle, is formed. If cured meat is omitted and nitrate, sucrose, and any ordinarysoluble proteins or peptones are added, no carbon dioxide forms. With somestrains of the bacilli, gas is formed if nitric-oxide hemoglobin or nitric-oxide hemo-chromogen is present in the sucrose-nitrate medium. Some strains of the genusBacillus do not form acid in the standard sugar-veal infusion broths (47) but doform acid and carbon dioxide when grown in nitrate sugar-cured porkmedium.This was observed with the disaccharides and one of the pentoses, xylose. In thecase of sucrose-nitrate-cured spiced ham carbon dioxide is formed as follows:The first reaction observed is the splitting of some of the disaccharides to mono-saccharides. Later, lactic acid is found in appreciable quantities and alsomuchcarbon dioxide. In the closed container, as used for canned meats, where there isno escape for acetaldehyde, much 2,3-butylene glycol is formed. The order ap-

174


mbr.asm

.org/D

ownloaded from



pears to be disaccharide to hexoses to triose compounds and then to pyruvic acid.A part of the pyruvic acid is reduced to lactic acid and a part is split into acetalde-hyde and carbon dioxide. The acetaldehyde may either be reduced to alcohol,oxidized to acetic acid, or condensed to form acetyl methyl carbinol which can bereduced to 2,3-butylene glycol.We have never observed the formation of carbon dioxide in media other than

nitrate-sugar-cured pork meat or nitric-oxide myohemoglobin or hemochromogenexcept in a few instances where thiamin was added to the nitrate-sugar medium.Presumably, phosphorylated vitamin B1 acts as a coenzyme which decarboxylatespyruvic acid; and there is a comparatively large amount of thiamin in pork.Thiamin is not a complete factor in this mechanism since gas was not observedto be formed by many strains of bacilli capable of gaseous fermentation in thenitrate-sucrose-cured pork medium (47).The spores of this group are killed by the present-day processing schedule for

canned spiced ham or luncheon meats. In the presence of 3 to 3.5% NaCl, 2.33oz (0.15%) NaNO3, and 0.125 oz (0.008%) NaNO2 per 100 pounds of meat, the6-pound cans may be inoculated with spores of clostridia, bacilli, or thermophilesand retorted at 112.8C (235F) for 3.5 hours so that an inside temperature of107.2C (225F) is reached with a finished product that will keep at temperaturesbelow 55.5C (132F).

ZoBell (116) has demonstrated the ability of some micro6rganisms to destroynitrites as they are produced from nitrates, and recommends that tests for ni-trates be made in conjunction with tests for nitrites when the latter substance isnot found. In this connection, we have observed that hydroxylamine may befound in the old-style, long-mixed cure for hams towards the end of the curingperiod (60 to 80 days). The present-day ham is cured in less than 20 days.Lindsey and Rhines (68) have demonstrated the production of hydroxylaminefrom the reduction of nitrates and nitrites by various bacteria. Hydroxylaminehydrochloride, when added to nutrient agar, inhibits the growth of food-poisoningvarieties of staphylococci in dilutions of 1:20,000, and many of the commonmolds in dilutions of 1:15,000. Kitasato (53) has reported that hydroxylaminewhen added to culture media inhibited the growth of Clostridium tetani, Clostri-dium chauvei, and Clostridium septicum.

5. ACTION OF SALT (NACL) ON BACTERIA

(a) Action of Salt on Bacteria in Meats. For the past half century the demandfor heavily salted foods has lessened in many parts of the world. Salt-preservedfish and meat were for a long time staple articles of food in world trade, and thepreservation of foods by salting has proved its efficacy since time immemorial(55). The New England-West Indies trade in salted meat was of importance incolonial times (99).

Lessening the amounts of salt used in food preservation has resulted in changesin microbiological flora and brought forth many phenomena of interest to thebacteriologist. Tanner and Evans (95) have discussed the literature on the effectof salt on pathogenic bacteria and stated that most investigators indicate that

175


mbr.asm

.org/D

ownloaded from


L. B. JENSEN

salt in the concentrations used in food preservation is not a bactericide but acts asa preservative by inhibiting many species of bacteria. Tanner and Evans con-clude from their own extensive experiments that salt is the most active preserva-tive used in meat-curing solutions.

Petterson (78) has shown that the preserving action of salt in meat and fish isnot sharply defined. Salt exhibited a selective action and did not inhibit allbacteria. Anaerobes were inhibited by 5% NaCl whereas aerobic rods, faculta-tive anaerobic rods, and micrococci were not greatly inhibited. The rod formswere more easily suppressed by salt than the cocci. Ten per cent NaCl appearedto inhibit most bacteria, alth'ough a few grew in media containing up to 15%NaCl. Various yeasts appeared to persist and grow in media containing over15% NaCl.At this point attention is called to the diverse results which can be observed

when, for instance, 5% salt is used in pickling meats or is added to liquid culturemedia. Tanner and Evans (95) have demonstrated that the inhibitory effect ofsalt on Clostridium botulinum is different when the same concentrations of NaClare used in the different culture media. Glucose agar containing 6.5% or more ofNaCl did not support growth. Nutrient broth containing 6.7% NaCl allowedone culture to show visible signs of growth and five strains of Clostridium botulinumto grow and produce toxin. It was necessary to add 7.8% NaCL to prevent thegrowth of all strains in nutrient broth. When 7.3% NaCl was used in glucosebroth and 7.8% in pork-infusion broth, growth and toxin formation took place.Growth took place in 10.5% NaCl in glucose broth and 12% was needed to inhibitgrowth in this medium. With cooked pork toxin formation was prevented by 5%NaCl.When over 3.5% NaCl is used in curing whole pieces and comminuted meats,

most anaerobes are suppressed although there are a few strains of nonpathogenic,sporogenous anaerobes that can germinate and multiply when given heat treat-ment several times greater than that necessary to destroy spores of Clostridiumbotulinum. Fortunately these strains are encountered very infrequently and atthis time are a curiosity in culture collections.

Bacillus foedans of Klein, which we have equated with Bacillus putidus ofWeinberg (48), is inhibited in hams containing 3% NaCl and undergoing thesmokehouse processing. This organism is very proteolytic and grows in non-pickled areas, sometimes formed through by-passing the bifurcation of the iliacartery during pumping. This condition may be differentiated from sarcosporidialactivity, which likewise causes a butyrous area, but is associated with a normalsalt content and normal organoleptic characteristics.Many species of aerobes and facultative anaerobes (as well as strict anaerobes)

can grow in high salt concentrations in brines which contain large pieces of variousanimal tissues; but the growth takes place at the interfaces of brine and tissuesand not readily in the clear brine. When meat infusions or whole blood are addedto 15 to 20% clear NaCl brines, the growth of salt-tolerant microorganisms is veryslow. When large pieces of meat are added to these vats and the salometer read--ings are kept constant, growth proceeds more rapidly, usually on the surfaces of

176


mbr.asm

.org/D

ownloaded from



tie meat. The mechanis of growth at the interfaces appear not to be wellunderstood at the present time.

In the curing of comminuted meats, the ratio of salt to total moisture obviouslyaccounts for the low quantities of salt which suffice to inhibit many micro-organisms. For instance, in composite beef muscle the water content is about71% and protein 18.7% with a water-to-protein ratio of 3.8, while in lean porkthe water content is 64.5% and protein 18.9% with a water-to-protein ratio of 3.4(74). A higher ratio of salt to water accounts for the preservative effect of saltin meats as compared to liquid culture media; but the problem of free and boundwater in tissues confronts one here as in so many other fields of food technology.Long experience has taught the curing departments of the industry the preciseamounts of salt necessary (in addition to the lawful amounts of nitrates andnitrites) to be effective in each of the many kinds of meat foods.

(b) Effect of Impurities in Salt. A phase of the salt curing of meat which hasreceived attention from time to time is the effect of impurities in the salt. Manycommercial meat processors are of the opinion that impurities in common salt(calcium, magnesium) affect the meat adversely, whereas some believe that moredesirable cures, from the standpoint of rapid penetration, are affected. Moultonand Lewis (75) made up sodium chloride curing solutions containing 5% CaCl2,or 1% CaCl2 + 1% MgSO4, or 1.5% CaCl2 + 0.5% MgSO4 for curing hams,butts, and beef knuckles. They found no consistent effects of these impurities insodium chloride brines upon the rate of penetration of the NaCl into the meat.They concluded that commercial salts would show no measurable differences inpenetration rates owing to the usual impurities.There is an extensive literature on the physiologically antagonistic effects of

ions (20). There is evidence that the toxic action of an ion can be nullified byanother different ion. In the case of bacteria there is a quantitative antagonismbut the qualitative effects of all cations may be alike. The toxic effect of a mono-valent salt like NaCl can be neutralized by the addition of a divalent salt insuitable proportion. Winslow and Falk (112) have found that 0.145 M CaCl2 +0.290 M NaCl was toxic to Escherichia coli, but a solution of 0.145 M CaCl2 +0.680 M NaCl was nontoxic.

Tressler and Murray (102) have noted that the presence of calcium and mag-nesium compounds as impurities in salt accelerated the development of the salt-fishy odor and taste of salted fish. It has been observed that if pure salt wasused for the salting of fish, the product when thoroughly freshened was nearly in-distinguishable from fresh fish. Tressler (101) had observed that impurities inthe salt exerted a depressing action on the permeability of the cell walls and thusslowed down the penetration of salt into the muscle tissues. The various effectsof salt impurities have been studied by E. Hess (28) who states: "Spoilage, asmeasured by rise in trimethylamine and bacterial content, of cod press-juice con-taining 21 grams salt per 100 ml. (about 80% saturated) is delayed longest withpure NaCl, and increasingly less with mined evaporated, mixed crude, Mediter-ranean and Turk's Island solar salts. This order corresponds to decreasing per-centages of NaCl and increasing percentages of impurities in these salts. The

177


mbr.asm

.org/D

ownloaded from


L. B. JENSEN

differences in the salt action increase with lower temperatures." Hess used fish-muscle press-juice instead of the muscle itself which eliminates the penetrationfactor and also makes bacterial counts more accurate. Hess quotes unpublisheddata of Gibbons who observed that a medium not containing calcium or mag-nesium ions (prepared from dialyzed drip of fish muscle, washed agar, and pureNaCl) did not support the growth of red halophilic bacteria, or only very poorly,whereas the addition of traces of calcium or magnesium or replacement of thesodium chloride by solar salt resulted in good growth. The same effects can beobserved on cells of red halophilic bacteria in the presence of CaCl2, MgCl2, andMgSO4 in high concentrations of NaCl. Beef cured in 1000 salometer brine withsolar sea-salts and stored at 32.2 to 37.8C (90 to lOOF) will spoil with character-istic trimethylamine odors after 5 months, showing a typical flora of Serratia,Achromobacter, and Micrococcus. The work of Tarr (97) is interesting in thisconnection in that he has discovered an enzyme, "triamineoxidease," existing incells of five different genera of bacteria which can form trimethylamine.The dry-curing process employed to cure country hams and southern-style

hams is interesting from the standpoint of salt permeability. In observing thispractice in many places in America, it was noted that the farmer or commercialprocessor first rubs sodium nitrate over the ham, usually rubbing from the sanktowards the butt. The shank (tibia and fibula) is "sawed long" to effect marrowsealing. The nitrate appears to increase permeability or "cause swelling and amoist condition." Then after a time salt or salt and sugar are rubbed into theham in the same manner as the nitrate. The process is repeated a number oftimes for about seven weeks, and then the hams are smoked for varying lengthsof time. The effect of nitrate on the permeability has not been given much sci-entific study (110), but the art of curing in this manner is very old. Surprisinglyfew pieces cured in this way show frank spoilage (as distinguished from incipient"souring").

It is known that very dilute nitric acid softens the connective tissue binding themuscle fibers. This action may aid in "tendering" certain cured meats. Studiesare needed on the action of nitrates, nitrites, salt, sugar, and enzymes on musclefibers, sarcolemma, sarcoplasm, myofibrils, discs, and other structures of muscle.Rockwell and Ebertz (86) conclude that the preserving effect of NaCl involves

more than its dehydrating capacity. For instance, if common salt inhibitsbacterial growth by means of dehydration, then other equally efficient dehydrat-ing salts should serve as inhibitors. Magnesium sulphate has greater dehydrat-ing,effect on proteins than NaCl but is not as efficient in preventing the growth ofStaphylococcus aureus. They conclude from their experiments that the preserv-ing action of NaCl on proteins involves more than dehydration, there being fourother factors operative. These factors are: the direct effect of the chloride ion,removal of oxygen from the medium, sensitization of the test organism to C02,and interference with the rapid action of proteolytic enzymes.

(c) Bacteria in Salt. From time to time, salt contaminations leading tospoilage have been suspected in meat processig, but no unimity of opinonexists either among food bacteriologists or operating men in the industry concern-

178


mbr.asm

.org/D

ownloaded from



ing the hazards of contamination in salt. There are even those food bacteri-ologists who doubt the existence of obligate halophiles as spoilage organisms, andwho view these bacteria as salt-tolerant forms. The distinction is not clearlymade in some of the literature on salt. The observations of Clayton and Gibbs(13), Robertson (85), and ZoBell, Anderson, and Smith (117) indicate thathalophilic bacteria are distinct species indigenous to environments containinghigh concentrations of sodium chloride. ZoBell et al. (117) found an average of167 obligate halophiles in Great Salt Lake water having a salinity of 27.6%.These halophiles required at least 13% salt for growth. These bacteria are be-lieved to have become acclimatized to increasing salt concentrations during thetime that the water of old Lake Bonneville evaporated to leave its saline remnant,Great Salt Lake.The studies of Stuart, Frey, and James (92) and Stuart and Swenson (93)

indicate that halophilic bacteria are adapted forms of ordinary bacteria known tobe indigenous to other environments, such as soil, etc. Lochhead's work (69) onhalophilic bacteria should be consulted in this connection.The data obtained during the past decade (37) from microbiological analyses of

mined salt and vacuum-pan salt of various grades indicate strongly the improba-bility of contaminations in salt sufficiently important to lead to meat spoilage.Certain of the solar sea-salts may, however, need careful scrutiny. Yesair (113),who examined many grades of salt, advised microbiological examinations of thesalt as a control measure in the food industry.The question of the nature and origin of halophiles has been reviewed elsewhere

(37).(d) Some Effects of Salt on Bacteria. Large initial numbers of contaminating

bacteria tend to grow in meats that are highly salted. The observation has beenmade many times that large numbers of non-halophilic or non-salt-tolerantbacteria tend to neutralize the inhibitory effect of salt. It appears that the largerthe inoculation the greater the salt tolerance of the bacteria. The work of Sher-man and Albus (89) shows that when active reproduction takes place there ismarked destruction of the cells in the presence of 5% NaCl. This observation isborne out in practical application where it is noted that fresh meats showing lightbacterial contamination keep better when salted quickly than when held for atime before salting. The old cells are not as sensitive to salt as the actively grow-ing cells.The effect of sodium chloride on thermal death times of bacteria has been

studied by Esty and Meyer (19) who found that NaCl in low concentrations(0.5 to 1%0) markedly increased the thermal resistance of spores of Clostridiumbotulinum. At 2% concentration of salt, this effect was lost. Up to 8%, littleor no effect of the salt was observed. Above this concentration, up to 20% NaClsolution, the effect was to decrease the thermal death times. Viljoen (106)studied the adjuvant effect of NaCl upon thermal death times of spores in pealiquor. He found a protective influence of salt in the range of 1 to 2.5% NaCl,and 3% salt shows about the same protection as 0.5%. Four per cent NaCl in-creases very slightly the destructive effect of heat.

179


mbr.asm

.org/D

ownloaded from


L. B. JENSEN

In the case of micrococci, salt appears to exert a p1:otective action against theeffects of heat. Alkaline solutions of salt reduce the thermal death times ofspores in a very striking manner.The terms "obligate halophile," "salt tolerant," "salt resistant," and "faculta-

tive halophile" may all be employed to indicate the characteristics of a strain,but there is still difference of opinion as to the distinction between such terms.If Kluyver and Bahrs' (54) doctrine of "physiological artifacts" is true, i.e., thathalophilism is developed by conditions of artificial culture in saline media from aprepotency latent in the cell of the saltless environment, much remains to be donebefore the terminology becomes clear. They demonstrated the interconverti-bility of nonhalophilic Microspirum desulphuricans (Beijerinck) and halophilicMicrospirum aestuarii (Van Delden) by gradual alteration of the salt content ofthe medium. They suggested that these forms are all derived from a commonparent strain, and hence the specific identity of the halophiles and non-halophiles.F. B. Smith has written an excellent review of this subject (90).There are many strains of obligate halophilic molds, usually divergent forms of

brown molds, that exhibit all degrees of salt tolerance. Some are psychrophiles,but most strains are mesophiles. Some resemble species of Torula, othersresemble species of Hemispora, Oospora, or Sporendonema. Aspergillus candiduwill grow on salted smoked meats and produce small reddish patches.

6. SOME RECENT DEVELOPMENTS IN MEAT TECHNOLOGY

The war has brought about some interesting developments in meat technologyas well as in food technology generally. The outstanding developments in theproduction of foods on a scale hitherto undreamed of have been along the lines ofdehydration of pork, beef, eggs, milk, soups, and some poultry meats. Boneless,well-trimmed beef (produced under bacteriological control) and other meats arefrozen; and through the efficient handling of the Quartermaster Corps and Veteri-nary Corps, these meats are made available for consumption in battle zones.The microbiology of freezing meats and of all kinds of stored fresh and curedmeats and frozen eggs has taken on new life. Bacteriological studies have shownthat there are critical storage temperatures at which the "indigenous" flora dieoff rapidly. For instance, we (42) have shown that frozen egg magma inqculatedwith several strains of Escherichia coli, several species of Pseudomonas, Achromo-bacter, Flavobacterium, and Micrococcus, when frozen, divided into two lots, andthen held at -22 F (-30 C) and 22 F, (-5.6 C) shows sharp differences in re-duction of all bacteria in four weeks. Strangely enough, the bacteria are reducedto very low numbers of viable cells at 22 F;and there is very little or no diminutionof viable cells to be found at -22 F. There are also critical holding temperaturesfor beef, pork, lamb, veal, and dressed poultry. Atmospheric oxidations compli-cate some of the benefits derived from the "higher" temperatures of storage.

(a) Dehydration of Meat. Processes for dehydration of meat of all kinds havecome to the fore during the war years, and the reader is referred to the excellentwork of von Loesecke (108) for details of the drying and dehydration of foods.Four kinds of meat have been prepared: fresh meat cooked and raw, and cured

180


mbr.asm

.org/D

ownloaded from



meat cooked and raw. Most of the dehydrated beef and pork of commerceLs pre-cooked fresh meat dried to 10% moisture and then packed and sealed in tin cans.Extensive studies carried out by the U. S. Department of Agriculture and themeat packers have shown that from a bacteriological standpoint dehydratedmeat, made in accordance with Federal specifications, is safe and will remain sowhen stored without refrigeration in hermetically sealed containers (57). Insuch dehydrated meat, we notice a rapid decline in the numbers of bacteria. ThepH is 5.8 to 6.2. Salmonella sp. staphylococci, Clostridium botulinum, and otherpathogens will not grow in this product until the meat is rehydrated to containover 30% moisture.Commercial desiccation of foods is no new thing. Du-ring the Civil War troops

were supplied with desiccated vegetables, soup mixtures, apples, peaches, etc.General Sherman wrote in his memoirs: "During the Atlanta campaign we weresupplied by our Commissaries with all sorts of patent compounds, such as desic-cated vegetables, and concentrated milk, meat biscuits and sausages, but some-how the men preferred the simpler and more familiar forms of food, and usuallystyled these 'desecrated' vegetables and 'consecrated milk'." Dehydrated ra-tions were used in the Boer War; and the A. E. F. veterans of 1917, who accountedfor the consumption of 10 million pounds of desiccated foods, exhibited about thesame attitude as their grandfathers towards these dried foods. Following WorldWar I the dehydration of foods was not extensive, but wvith the coming of WorldWar II this business has taken on new life. Improved methods may save theindustry from complete collapse after the war ends.An interesting application of rapid dehydration of meat has been employed by

Ritchell, Piret, and Halvorson (84) to form products like dry or summer sausage.They investigated air-drying of uncooked cured meats. In contrast to the usualsausage drying operations, shorter tinmes of drying were necessary to obtain aproduct containing 25 to 30% moisture.Work done by Dr. E. E. Rice and Dr. H. E. Robinson of the Research Labora-

tories of Swift & Company shows that:

Protein quality is not significantly reduced during dehydration or canning unless dietsvery low in proteins are considered.Vitamin retention of pork and beef undergoing dehydration or canning is similar to that

for household cooking of similar meats, being thiamin, 60 to 70%; riboflavin, 90 to 100%;niacin, 90 to 100%; and pantothenic acid, 70 to 80%.Cured pork undergoing commercial canning (12-oz. can) retains 67 per cent of its thiamin,

90 per cent of its riboflavin, 94 per cent of its niacin, and 76 per cent of its pantothenic acid.Thiamin retention in 6-lb. cans is lower, being 40 to 50%.During storage of either canned pork, dehydrated pork or dehydrated beef at tempera-

tures up to 99 F (37.2 C) there is little or no loss of niacin, riboflavin, or pantothenic acidover a period of 219 days. Above 120 F (48.9 C) there are slow losses of riboflavin and panto-thenic acid. Thiamin decreases more rapidly, showing some loss at 80 F (26.7 C). After 293days' storage the thiamin retention in canned pork is 52 per cent. In dehydrated pork theretention is poorer, being28per cent after219 days at80 F (26.7 C). At higher temperaturesthere is almost complete destruction of thiamin in both products.

Molds can develop on dehydrated meats when these foods are exposed to theatmosphere and stored at relative humidities below 75% in the temperature range

181


mbr.asm

.org/D

ownloaded from


L. B. JENSEN

of 10 to 37.2 C (50 to 99 F). Molding is, however, not a hazard in the tins andother containers used in distribution of this product.

(b) Bacterial Standards. The bacteriologi6al control of foods by the criteria ofagar-plate counts has long been a "standard method" for the inspector and pro-ducer. Most bacteriologists have been confronted with the dilemma of evaluat-ing a food product from the "count" or the more laborious examination.The subject of bacterial oounts, i.e., bacteria growing into colonies in agar

plates when incubated in the range 20 to 37 C (68 to 98.6 F) for varying lengthsof time, as criteria for judgment of many foodstuffs is controversial at the presenttime. One school of thought would throw over the existing agar-plate countstandards for most foods and replace this method by qualitative bacteriologicalexaminations together with organoleptic criteria and chemical examinations.This group claims that when, let us say, a food like dried eggs contains 500,000"viable aerobes" per gram and the "standard limits" are 250,000 per gram, theegg powder should not be rejected as inedible. There is undoubtedly much to besaid for this position. However, the regulatory laboratories must have somecriteria and limits of numbers if their investigators are to rely on "standards ofnumbers of bacteria permissible." The total number of bacteria growing on agarplates do, in many instances, serve to guide the processing of foods so that in-cipient spoilage does not result.

In meat foods, for the most part, it is more important to know both the kindsand numbers of the flora present than to know merely the numbers of micro-organisms. Because most bacteria which grow in meat under refrigeration arepsychrophiles or facultative psychrophiles, such as many species of Achromobacter,Pseudomonas, or Serratia, it may be considered that a "high count" does notnecessarily indicate that the food is unsound if the organoleptic characteristics aresatisfactory. Again, if counts are made on certain sausages, such as Thuringer,the number of lactobacilli found is very great indeed (40).

In the preparation of frankfurters, bologna, meat loaves, and other com-minuted meat foods, the bacteriologist must be guided by qualitative bacteri-ological data. The total agar-plate count cannot presage the possibility ofoxidizing effects leading to discolorations.Meat, when produced under Federal inspection, is not likely to harbor patho-

gens, according to the data of the Bureau of Animal Industry, the Meat Inspec-tion Division, or the Research Laboratories of the Meat Industry. There are no"normal" plate counts for meats in the literature available to us. A satisfactorycount on meats may depend upon whether or not the meat is destined for use insausage or, in the case of beef, whether or not it is to be aged in coolers for sometime to induce tenderness, flavor, and juiciness. As there are several hundredkinds of meat foods produced continuously, no simple answer is forthcoming inrespect to a "single standard" for these foods. It would be helpful if we couldstate with conviction founded upon experience that liver sausage should not ex-ceed sixty thousand aerobes per gram or that pork links or patties should notexceed several hundred thousand aerobes per gram. However, perfectly ediblesausage might show three times these figures, depending upon the length of timethe food has been stored in the shop or commissary refrigerators.

182


mbr.asm

.org/D

ownloaded from


MiCROBIOLOGY AND PRESERVATION OF MEATS

It would appear that the ideal laboratory examinations of a finished foodproduct should disclose the presence or absence of pathogens or toxins, and thatboth plate counts and determination of flora may be used in control of theprocessing. Methods for direct microscopical counts of foodstuffs are not want-ing in number. Some inherent difficulties in direct-count methods are: (a)numbers less than 1 X 105 organisms per gram in food substances, such as muscle,cannot be accurately counted; (b) dead cells are counted (this is not a seriouserror); (c) errors in sampling, preparation, grinding, smearing, etc., are noteliminated; (d) "primitive" forms are not detected (41), nor are types determinedexcept in a very general manner. Other shortcomings are perhaps apparent tothe analyst, but in theory the method of direct count obviously has mtuch to com-mend it. However, the problem is not considered impossible of solution, and thedirect count could supplant the agar-plate count in many places.

(c) Hams. New applications of old methods of establishing bacteriologicalcriteria for the curing and smoking of hams (not tenderized, not ready-to-eat)have been effected and incorporated into official specifications. These applica-tions are based on the bacteriological observations showing that cured hamsshould be smoked at temperatures below 125 to 130 F (51.7 to 54.4 C) for severaldays for the smoke to penetrate well, that some dehydration should occur, andabove all, that the tissues do not become denatured through heat coagulation.It is well known to the bacteriologist that coagulated proteins, such as cookedmeats or boiled eggs, are more vulnerable to enzyme action, and bacteria find amore suitable substrate for growth on them than on raw substances. Hams andshoulders, when tenderized (partial cooking), or cooked in the smokehouse so thatthey are ready to eat, are to be considered in the same category as comminuted,cured domestic sausage products which must always be kept under adequate re-frigeration. It has been showm (43) that cooked hams, cooked poultry andstuffings, sandwiches prepared with ham, eggs, fish, and fowl, and soup stocksshould never be held in the "incubation zone" of staphylococci longer than fourhourg if gastrointestinal irritation due to staphylococci is to be prevented. Theincubation zone is considered to be from 60 to 115 F (15.6 to 46.1 C), and as asafety margin for institutional and military mess halls the range from 50 to 120 F(10 to 52.9 C) is advocated.

(d) Control of Spores in Canned Meats. The heating schedules for cannedmeats developed through the stimulus of war have been based largely upon ex-isting data derived from studies of spores. The direction of the work in thepractical canning of spiced ham, luncheon meats, and sausage products is toretort the canned food according to a "botulinum schedule." Many meat foodssubjected to this temperature would not be very palatable; hence, the need aroseto produce a canned meat that would neither spoil nor present a health hazard.Also high-temperature processing for long periods lessens the vitamin content ofmeat foods. The thiamin and pantothenic acid content, especially, may be re-duced, although niacin and riboflavin are not significantly reduced. When3.5% NaCl, 2.33 oz. (0.15%) NaNO3, and 0.125 oz (0.008%) NaNO2 in each 100pounds of comminuted meat are used, and the cured meat is packed in 6-poundcans and retorted for 3.3 hours so that the inside temperature reaches 225 F

183


mbr.asm

.org/D

ownloaded from


L. B. JENSEN

(107.2 C), the processed cans of meat, even though seeded with spores of Clostri-dium sporogenes and of Bacillus sp., will keep at any temperature below 132 F(55.6 C) for a time depending upon the rate of production of hydrogen from thechemical action of the contents upon the metallic can (hydrogen swells).As much as 22% soya flour or "meat extender" can be added to comminuted

meats and canned according to this schedule of curing and heating with the re-sulting product stable under expected temperatures (132F [55.6 C] and lower).Tests were conducted by Jensen and Hess (49, 50) who inoculated soya flour tocontain over one million spores of gas-forming, anaerobic thermophiles per gram.Thirty 6-pound cans of comminuted luncheon meat (pork and beef) containing22% of this soya flour were incubated for 9 months at 132 and 99 F (55.6 and37.2 C) and found to be stable. The control cans without salt and nitrate swelledand burst in a few days at these incubation temperatures.The "bombage" of canned meat foods stored without refrigeration in the

tropics has been caused by viable spores of Bacillus sp. These spores are not in-hibited from germination by 3.5% NaCl with sodium nitrate. These sporesmust be killed to produce a stable canned pork or luncheon meat. Spores ofthermophiles may remain dormant in canned meat for long periods of time.Recently, Wilson and Shipp (111) examined bacteriologically a can of roastedveal, packed for the explorer Parry in 1824, and recovered six strains of sporingbacilli. The strains grew best at 37 C, all strains grew well at 55 C, and some grewat 60 C. The survival of spores in this can of veal in viable condition for over acentury appears to have no parallelPin bacteriological records.

REFERENCES1. ADAMI, J. G. 1899 On latent infection and subinfection, and on the etiology of

hemochromatosis and pernicious anemia. J. Am. Med. Assoc., 33, 1509-1514, 1572-1576.

2. ALLEN 1932 Comnmercial Organic Analyses. Fifth ed., 9, 275-276. P. Blakiston'sSons & Co., Philadelphia, Pa.

3. ARNOLD, L. 1928 The passage of living bacteria through the wall of the intesti.e andthe influence of diet and climate upon intestinal auto-infection. Am. J. Hyg., 8,604-632.

4. BARNARD, R. D., AND GOWEN, G. H. 1932 Greenish discoloration produced on bloodagar by the growth of pneumococcus. Proc. Soc. Exptl. Biol. Med., 29, 521-524.

5. BITTENBENDER, W. A., DEGERING, E. F., TETRAULT, P. A., FEASLEY, C. F., ANDGWYNN, B. H. 1940 Bactericidal properties of commercial antiseptics. Ind.Eng. Chem., 32, 996-998.

6. BLUM, H. F. 1941 Photodynamic action and diseases caused by light. Chap. 9.A.C.S. Monograph, New York, N. Y.

7. BOXER, S. 1906 Ueber das Verhalten von Streptokokken und Diplokokken aufBlutnihrboden. Centr. Bakt., Parasitenk., Abt I, Orig., 40, 591-600.

8. BOYER, E. A. 1926 A contribution to the bacteriological study of ham souring. J.Agr. Research, 33, 761-768.

9. BROH-KAHN, R. H., AND MIRSKY, I. A. 1938 Studies on anaerobiosis. The nature ofthe inhibition of growth of cyanide-treated E. coli by reversible oxidation-reductionsystems. J. Bact., 35, 455-475.

10. BROOKS, J., HAINES, R. B., MoRAN, T., AND PACE, J. 1940 The function of nitrate,nitrite and bacteria in the curing of bacon and hams. Dept. Sci. Ind. Research,Food Invest., Special Rept. 49, 2-4, London.

184,


mbr.asm

.org/D

ownloaded from



11. BURN, C. G. 1934 Postmortem bacteriology. J. Infectious Diseases, 54, 395-403;Experimental studies of postmortem bacterial invasion in animals. Ibid., 388-394.

12. CANFORA, M. 1908 Ueber die Latenz der Tetanussporen im tierischen Organismus.Centr. Bakt., Parasitenk., 45, 495-501.

13. CLAYTON, W., AND GIBBS, W. E. 1927 Examination for halophilic micro-organisms.Analyst, 52, 395-397.

14. D]SAN, H. K. 1938 Utilization of Fats, 186-194. Chemical Publishing Co., New YorkCity.

15. EWELL, A. W. 1940 The tenderizing of beef. J. Am. Soc. Refrig. Eng., April,237-240.

16. EWELL, A. W. 1942 Production, concentration and decomposition of ozone by ultra-violet lamps. J. Applied Phys., 13, 759-767.

17. EWELL, A. W. 1942 Cutting shrinkage losses in retail meat coolers. The NationalProvisioner, April 11, 1942.

18. EWELL, A. W. 1942 Conservation of refrigeration during wartime. Refrig. Eng.,September, 1942.

19. ESTY, J. R., AND MEYER, K. F. 1922 The heat resistance of the spores of B. botulinusand allied anaerobes. XI. J. Infectious Diseases, 31, 650-663.

20. FALK, I. S. 1923 The r6le of certain ions in bacterial physiology. Abstracts Bact.,7, 33-50.

21. GIBBONS, N. E., AND REED, G. B. 1930 The effect of autolysis in sterile tissues onsubsequent bacterial decomposition. J. Bact., 19, 73-88.

22. GREEN, D. E., AND PAULI, R. 1943 The antibacterial action of the xanthine oxidasesystem. Proc. Soc. Exptl. Biol. Med., 54, 148-150.

23. GRINDLEY, H. S., MACNEAL, W. J., AND KERR, J. E. 1929 The influence of potassiumnitrate on the action of bacteria and enzymes. Chap. 9, 2, 359. Studies in Nutri-tion, University of Illinois, Urbana, Ill. (Complete results in five volumes.)

24. HAGAN, W. A. 1925 The green coloration by certain streptococci on blood agar. J.Infectious Diseases, 37, 1-12.

25. HALDANE, J. 1901 The red colour of salted meat. J. Hyg., 1, 115-122.26. HART, P. D'ARcy, AND ANDERSON, A. B. 1933 The formation of green pigment from

haemoglobin by the pneumococcus. J. Path. Bact., 37, 91-105; the discolouration ofheated blood agar by streptococci. Ibid., 334-335.

ANDERSON, A. B., AND HART, P. D'ARcy 1934 The viridans effect of the streptococciand the production of the green pigment from haemoglobin by other reducing Sys-tems. Ibid., 39, 465-479.

27. HEKTOEN, L., ROBSCHEIT-ROBBINS, F. S., AND WHIPPLE, G. H. 1928 The specificprecipitin reaction of the muscle hemoglobin of the dog. J. Infectious Diseases,42, 31-34.

28. HEss, E. 1942 Studies on salt fish, VIII. J. Fisheries Research Board Can., 6,(1), 1-23.

29. HOAGLAND, R. 1914 Coloring matter of raw and cooked salted meats. J. Agr. Re-search, 3, 211-226.

30. HOAGLAND, R., MCBRYDE, C. N., AND POWICK, W. C. 1917 Changes in fresh beefduring cold storage above freezing. U. S. Dept. Agr. Bull 433.

31. HOLMAN, W. L. 1916 The classification of streptococci. J. Med. Research, 34,377-387.

32. HOPPE-SEYLER 1871 Quoted from Wells, H. G., 1918. Chemical Pathology, 82.W. B. Saunders Co., Philadelphia, Pa.

33. HtJLPHERS, G. 1933-34 Skallvatten floran i Svinlungan. Nordiska Veterina`rm6tet,800-813. Helsingfors, Finland.

34. INGRAM, M. 1939 The endogenous respiration of Bacillus cereus, II. J. Bact., 38,613-629.

35. JAcOBY, M. 1900 Ueber die fermentative Eiweisspaltung und Ammoniakbildung inder Leber. Z. physiol. Chem., 30, 149-174; 1901. Ueber autolyse der Lunge. Ibid.,

185


mbr.asm

.org/D

ownloaded from


33, 16130; 1903. Zur Frage der specifischen Wirkung- der intracellularen Fer-ments. Beitr. Chem. Physiol. (Hofmeister's), 3, 446-450.

36. JENSEN, L. B. 1942 Microbiology of Meats, 18-24. The Garrard Press, Champaign,Ill.

37. JENSEN, L. B. 1942 Ibid., 211-219.38. JENSEN, L. B. 1942 Ibid., 7-9; 155-163; 185-188.39. JENSEN, L. B. 1942 Ibid., Chapter 5.40. JENSEN, L. B. 1942 Ibid., 10, 17, 165.41. JENSEN, L. B. 1942 Ibid., 229.42. JENSEN, L. B. 1943 Bacteriology of ice. Food Research, 8, 265-272.43. JENSEN, L. B. 1944 Prevention of food poisoning by food preservation methods.

J. Am. Vet. Med. Assoc., CIV, No. 802, 63-65.44. JENSEN, L. B., AND GRETTIE, D. P. 1933 Action of microorganisms on fats. Oil and

Soap, 10, 23-32.45. JENSEN, L. B., AND GRETTIE, D. P. 1937 Action of microorganisms on fats. Food

Research, 2, 97-120.46. JENSEN, L. B., AND HESS, W. R. 1941 Action of nitrates on bacteria in cured meats.

Food Manuf., 16, 157-167. London, England.47. JENSEN, L. B., AND HESs, W. R. 1941 Fermentation in meat products by the genus

Bacillus. Food Research, 6, 75-83.48. JENSEN, L. B., AND HESS, W. R. 1941 A study of ham souring. Food Research, 6,

273-326.49. JENSEN, L. B., AND HESS, W. R. 1943 Control of thermophilic bacteria in canned

meat mixtures. Food Industries, 15, 66-68.50. JENSEN, L. B., AND HESS, W. R. 1943 Comprobacion de la Bacteria Termofila en las

Mezelas para Carnes Frias. La Maquina, 6, 16-19.51. JENSEN, L. B., AND URBAIN, W. M. 1936 Bacteriology of green discoloration in meats

and spectrophotometric characteristics of the pigments involved. Food Research,1, 263-273.

52. KILMER, F. B. 1901 The story of the papaw. Am. J. Pharm., 73, 272-285.53. KITASATO, S., UND WEYL, T. 1890 Zur Kenntniss der Anaeroben. Z. Hyg. Infek-

tionskrankh., 9, 97-102.54. KLUYVER, A. J., AND BAHRS, A. 1931 Konink. Akad. Wetenschappen, Amsterdam,

Proc., 35, 370.54. KOLLER, RAPHAEL 1938 Salt in the History and Culture of Mankind. Hallein,

Austria.56. KoSER, S. A., AND MCCLELLAND, J. R. 1917 The fate of bacterial spores in the animal

body. J. Med. Research, 37, 259-268.57. KRAYBILL, H. R. 1943 Dehydration of meat. Ind. Eng. Chem., 35, 46-50.58. LEA, C. H. 1938 Rancidity in edible fats. Dept. Sci. Ind. Research, Food Invest.,

Special Rep. No. 46 (London).59. LEA, C. H. 1939 The deterioration of fats in foods. Chemistry & Industry, 58,

479-484, (London).60. LEIFSoN, E. 1931 Bacterial spores. J. Bact., 21, 331-356.61. LEMBERG, R. 1935 Transformation of haemins into bile pigments. Biochem. J., 29,

1322-1336.62. LEMBERG, R., CORTIS-JONES, B., AND NoERiE, M. 1937 An oxyporphyrin haematin

compound as intermediate between protohaematin and verdohaematin. Nature,140, 65-66.

63. LEMBERG, R., CoRTIS-JoNES, B., AND NORRIE, M. 1938 Coupled oxidation of ascorbicacid and haemochromogens. Biochem. J., 32, 149-170; 171-186.

64. LEMBERG, R., AND LEGGE, J. W. 1943 Liver catalase. Biochem. J., 37, 117-127.65. LEMBERG, R., AND LEGGE, J. W., AND LocKwoOD, W. H. 1939 Coupled oxidation of

ascorbic acid and haemoglobin. Biochem. J., 33, 754-758.66. LEMBERG, R., AND LOCKWOOD, W. H., AND LEGGE, J. W. 1941 Studies on the forma-

186 L. D. JIENSEN


mbr.asm

.org/D

ownloaded from



tion of bile pigments from choleglobin and verdohaemochromogen and on theirisolation from erythrocytes. Biochem. J., 35, 363-379.

67. LEMBERG, R., AND WYNDHAM, R. A. 1937 Some observations on the occurrence ofbile pigment hemochromogens in nature and on their formation from haematin andhemoglobin. J. Proc. Roy. Soc. N. S. Wales, 70, 343.

68. LINDSEY, G. A., AND RHINES, C. M. 1932 The production of hydroxylamine by thereduction of nitrates and nitrites by various pure cultures of bacteria. J. Bact.,24, 489-492.

69. LOCHHEAD, A. G. 1934 Bacteriological studies on the red discolorations of saltedhides. Can. J. Research, 10, 275-286.

70. MCCULLOCH, E. C. 1936 Disinfection and Sterilization, 491. Lea and Febiger,Philadelphia, Pa.

71. McLEoD, J. W., AND GORDON, J. 1922 Production of hydrogen peroxide by bacteria.Biochem. J., 16, 499-506.

72. MCLEOD, J. W., AND GORDON, J. 1925 Further indirect evidence that anaerobes tendto produce peroxide in the presence of oxygen. J. Path. Bact., 28, 147-153.

73. MEYER, K. F. 1936 Latent infections. J. Bact., 31, 109-135.74. MOULTON, C. R. 1926 Some factors affecting the water content'of sausage. Am.

Meat Inst., Chicago, Ill.75. MOULTON, C. R., AND LEWIs, W. Lee, 1940. Meat Through the Microscope. Second

ed., 250-256. University of Chicago, Chicago, Ill.76. NIELL, J. M., AND HASTINGS, A. B. 1925 The influence of the tension of molecular

oxygen upon certain oxidations of hemoglobin. J. Biol. Chem., 63, 479-492.77. NORRIS, C., AND PAPPENHEIMER, A. M. 1905 A study of pneumococci and allied

organisms in human mouths and lungs after death. J. Exptl. Med., 7, 450-472.78. PETTERSON, A. 1899 Experimentelle Untersuchungen uber das Conserviren von

Fisch und Fleisch mit Salzen. Berlin. klin. Wochschr., 36, 915; 1900, Arch. Hyg., 37,171-238.

79. QUASTEL, J. H., AND WOOLDRIDGE, W. R. 1927 The effects of chemical and physicalchanges in environment of resting bacteria. Biochem. J., 21, 148-168.

80. RAMSBOTTOM, J. M., AND RINEHART, C. A. 1940 Fruit enzymes. Food Industries,12 (June), 45-47.

81. REEVES, J. R., AND MARTIN, H. E. 1936 The r6le of bacteria in autolyzing tissue.J. Bact., 31, 191-202.

82. REITH, A. F. 1926 Bacteria in the muscular tissues and blood of apparently normalanimals. J. Bact., 12, 367-383.

83. RICHARDSON, W. D. 1907 Nitrates in vegetable foods, in cured meats and elsewhere.J. Am. Chem. Soc., 29, 1757-1767.

84. RITCHELL, E. C., PIRET, E. L., AND HALVORSON, H. 0. 1943 Drying of meats: Rateof dehydration of uncooked cured ground meats. Ind. Eng. Chem., 35, 1189-1195.

85. ROBERTSON, MADGE E. 1931 A note on the cause of certain red colorations on saltedhides, etc.... J. Hyg., 31, 84-95.

86. ROCKWELL, G. E., AND EBERTZ, E. G. 1924 How salt preserves. J. Infectious Dis-eases, 35, 573-575.

87. RUEDIGER, G. F. 1906 The cause of green coloration of bacterial colonies in blood-agar plates. J. Infectious Diseases, 3, 663-665.

88. SALKOWSKI, E. 1890 Ueber Autodigestion der Organe. Z. klin. Med., 17, Supp.77-100.

89. SHERMAN, J. M., AND ALBUS, W. R. 1924 The function of lag in bacterial cultures.J. Bact., 9, 303-305.

90. SMITH, F. B. 1938 The determination of halophilic vibrios (N. spp.) Proc. Roy. Soc.Queensland, Brisbane, 49, 29-52.

91. STEPHENSON, M. 1939 Bacterial Metabolism, 57. Longmans, Green and Co., NewYork, N. Y.

92. STUART, L. S., FREY, R. W., AND JAMES, L. H. 1933 U. S. Dept. Agr. Tech. Bull. 383.

187


mbr.asm

.org/D

ownloaded from


L. B. JENSEN

93. STUART, L. S., AND SWENSON, T. L. 1934 Some new morphological and physiologicalobservations on salt tolerant bacteria. J. Am. Leather Chemn. Assoc., 28, 142-158.

94. TANNER, F. W. 1932 Microbiology of Foods, 482. Twin City Printing Co., Cham-paign, Ill.

95. TANNER, F. W., AND EVANS, F. L. 1933 Effect of meat curing solutions on anaerobicbacteria. Zentr. Bakt., Parasitenk., Abt. II, 88, 44-54.

96. TAROZZI, G. 1906 Ueber das Latentleben der Tetanussporen im tierischen Organis-mus und uber die Moglichkeit das sie einen tetanischen Prozess unter dem Einflusstraumatischer und nekrotisierender Ursachen hervorrufen. Centr. Bakt., Abt. 1.orig., 40, 305-311; 451-458.

97. TARR, H. L. A. 1940 Specificity of triamineoxidease. J. Fisheries Research Board,Can., 5 (2), 187-196.

98. TARR, H. L. A. 1941 The bacteriostatic action of nitrites. Nature, 147, 417-418;The action of nitrites on bacteria. 1942 J. Fisheries Research Board Can., 6,74-89.

99. THOMPSON, JAMES WESTFALL 1942 History of livestock raising in the United States,1607-1860. U. S. Dept. Agr., Agr. History Series No. 5.

100. TOPLEY, W. C., AND WILSON, G. S. 1938 The Principles of Bacteriology and Immu-nity. Second ed. William Wood and Co., Baltimore, Md.

101. TRESSLER, D. K. 1920 Some considerations concerning the salting of fish. U. S.Bur. Fisheries Documents, 884, 1-55.

102. TRESSLER, D. K., AND MURRAY, W. T. 1932 How brining with pure salts improvesfillets. Fishing Gaz., 49, No. 2, 1-3.

103. URBAIN, W. M., AND JENSEN, L. B. 1940 The heme pigments of cured meats. I.Preparation of nitric oxide hemoglobin and stability of the compound. Food Re-search, 5, 593-606.

104. VALENTINE, E. 1926 Differences in peroxide production and methemoglobin forma-tion of green (alpha) streptococci. J. Infectious Diseases, 39, 29-47.

105. VAN BRUGGEN, J. T., REITHEL, F. J., CAIN, C. K., KATZMAN, P. A., AND DoIsy, E. A.1943 Penicillin B: Preparation, purification, and mode of action. J. Biol. Chem.,148, 365-378.

106. VILJOEN, J. A. 1926 Heat resistance studies. 2. The protective effect of sodiumchloride on bacterial spores heated in pea liquor. J. Infectious Diseases, 39, 286-290.

107. VON FYRTH, OTTO 1916 Physiological and Pathological Chemistry of Metabolism,77. J. B. Lippincott Co., Philadelphia, Pa.

108. VON LOESECKE, H. W. 1943 Drying and Dehydration of Foods. Reinhold Publish-ing Corp., New York, N. Y.

109. WELLS, H. G. 1925 Chemical Pathology. Fifth ed., Chap. III. W. B. SaundersCo., Philadelphia, Pa.

110. WILSON, J. A. 1928 The Chemistry of Leather Manufacture, 165-166. ChemicalCatalog Co., New York, N. Y.

111. WILSON, G. S., AND SHIPP, H. L. 1939 Historic Tin Foods. Intern. Tin ResearchDevelopment Council, Publication 85, 49-55. Fraser Road, Greenford, Middlesex,England.

112. WINSLOW, C.-E. A., AND FALK, I.S. 1923 Studies on salt action, VIII, IX. J. Bact.,8, 215-236, 237-244.

113. YESAIR, J. 1930 Canning Trade, 52, 112-115; Bull. Nat. Canners' Assoc., Washing-ton, D. C.

114. YESAIR, J., AND CAMERON, E. J. 1942 Effect of curing agents on anaerobic spores.National Canners' Association. Washington, D. C.

115. ZINSSER, H., AND BAYNE-JONES, S. 1939 Textbook of Bacteriology. Eighth ed.,317, 934. D. Appleton-Century Co., New York City.

116. ZoBELL, C. E. 1932 Factors influencing the reduction of nitrates and nitrites bybacteria in semisolid media. J. Bact., 24, 273-281.

117. ZoBELL, C. E., ANDERSON, D. Q., AND SMITH, W. W. 1937 The bacteriostatic andbactericidal action of Great Salt Lake water. J. Bact., 33, 253-262.

188


mbr.asm

.org/D

ownloaded from


MICROBIOLOGICAL PROBLEMS MEATSvarious bacteria which can bring about these oxidizing reactions are:...

Documents

Transcript of MICROBIOLOGICAL PROBLEMS MEATSvarious bacteria which can bring about these oxidizing reactions are:...